The discovery of RNA interference (RNAi), a highly conserved post-transcriptional mechanism of gene silencing, promises to revolutionize medicine due to its potential to treat genetic, epigenetic and infectious disease. Efforts to unleash this potential, however, have met with challenges in delivery which have proven to be the major stumbling block for translation to humans. Recently we have discovered that bacteria encoding RNAi can induce potent gene silencing in mammalian cells (termed transkingdom RNAi, tkRNAi) in vitro and in vivo. This fundamental observation not only has significant implications for the interaction between the microbiome and host cells in vivo, but also offers a clinically compatible means to deliver RNAi in vivo for gene function research and for treating human diseases. This novel approach offers several advantages. Foremost is clinical safety since bacteria do not integrate genetic material into the human genome. tkRNAi also eliminates the need to chemically synthesize siRNA, and may mitigate host immune responses since the silencing RNA are produced inside target cells. Furthermore, the need for attenuation of bacteria, and the risk of environmental release of modified or mutated bacterial vectors can be addressed by technologies already developed for bacteria-based interventions. To translate this basic science observation toward clinical application, we have engineered non-pathogenic E. coli or attenuated Salmonella typhimurium to encode RNAi against beta-catenin, and demonstrated that oral administration of these bacteria can mediate potent and specific gene beta-catenin silencing in intestinal epithelial cells and has significant efficacy for treating polyposis in APCmin/+ mice. We further designed an optimized tkRNAi-based therapy for treating Familial Adenatomous Polyposis (FAP) patients. In collaboration with industry, this tkRNAi therapeutic has received FDA clearance for phase Ia/II trials. This is the first oral gene silencing therapy to be cleared by FDA, and also the first gene targeted therapy for FAP patients. This proposal is designed to elucidate and characterize the molecular processes and mechanisms of transkingdom gene silencing. Specifically, we will examine our hypothesis that bacteria-mediated RNAi can silence a target gene effectively and safely in mammalian cells sufficient for correcting disease phenotypes. We will use APCmin/+ mice as our in vivo model. To test this hypothesis, we will: 1) investigate the molecular processes and mechanism of transkingdom gene silencing; 2) comprehensively analyze the molecular mechanism of tkRNAi-mediated silencing of beta-catenin in the intestine, and 3) examine the possibility of developing a next generation tkRNAi technology. The successful completion of these studies will not only significantly enhance our understanding of interactions between the microbiome and host cells, but also be critical for developing and employing a tkRNAi-based therapeutic to meet urgent unmet medical needs of FAP patients and for using transkingdom gene silencing for treating other human diseases.